Identification of Significant Pathogenic Nontuberculous Mycobacteria Species from Presumptive TB Patients Using Partial hsp65 Gene Sequencing

Introduction

The Mycobacterium genus encompasses various species, including the Mycobacterium tuberculosis complex, which is responsible for tuberculosis (TB) infections, Mycobacterium leprae, which associated with leprosy, and a group of nontuberculous mycobacteria (NTM) species.¹ The NTM species, also known as Mycobacteria other than tuberculosis (MOTT), can be widely found in the common environment, such as soil and water. To this day, over 200 species of NTM are reported. They can be classified into slow-growing mycobacteria (SGM), which the colony formation requires at least seven days and rapid-growing mycobacteria (RGM), which form colonies in less than seven days.² As they are widely present in the environment, humans can be infected with NTM through regular activities.

There has been a significant global increase of opportunistic infection due to NTMs since the 1990s, resulting them emerge as important human pathogens.¹ However, unlike TB, diseases associated with NTM infections are not monitored by public health authorities; therefore, the true incidence of NTM infections may be underestimated, despite a steady rise in their prevalence. NTMs can cause infections in a wide variety of body sites. While approximately 90% of NTM infections frequently causes pulmonary diseases, there are also NTM-related skin diseases, lymphadenitis, and disseminated diseases.³ NTM pulmonary disease (NTM-PD) shares similar symptoms to TB, often leading to misdiagnosis and treatment failures. Furthermore, the NTM infections place some groups at higher risk, such as patients with chronic pulmonary diseases and immunocompromised conditions (for example, patients with HIV and cystic fibrosis).⁴ Pulmonary NTM infections are most commonly caused by Mycobacterium avium complex (MAC), Mycobacterium kansasii, Mycobacterium abscessus complex (MABC) and Mycobacterium chelonae.⁵

Currently, diagnostic modalities for identifying NTM infections are very limited. In Indonesia, the NTM infections are typically diagnosed using chest x-ray, liquid cultures, and differential diagnosis of MTB (eg, MPT64 antigen test).⁶ However, these techniques only allow to identifying NTM groups, rather than species specific. Generally, NTMs display initial resistance to the first line of the anti-TB drug. Their treatment requires different approaches from anti-TB drug, despite sharing similar symptoms. Moreover, the progression and disease characteristics are completely different.⁷ Furthermore, due to variations in treatment options among NTM species, it is necessary to identify the species and determine an appropriate regimen for the disease.⁸ Due to these limitations, studies on developing better diagnostic tools and treatments for NTM-related diseases are necessary.

In this study, an alternative approach for identifying mycobacteria was established through heat shock protein 65 (hsp65) partial gene sequencing, and the identification of NTM species can be done by sequence-based analysis. Additionally, our objective was to investigate the prevalence rate of each identified NTM species among the confirmed NTM infection based on the obtained result.

Materials and Methods

Selection of NTM Isolates

The samples used in this research were selected from retrospective samples in the form of stored isolates characterized as NTM suspects. All samples involved in the study categorized as “NTM suspects” were obtained from previous clinical research in which the inclusion is patient-based, and sputum sampling was carried out twice. For this research, samples underwent a thorough data screening process, ensuring the availability of relevant information such as demographic data, confirmation of positive Acid-Fast Bacilli (AFB) result through microscopic examination, negative result of MPT64 antigen test to differentiate between MTBC and NTM, and formation of cord factor in liquid bacterial culture. All stored isolates were sourced from a sample biobank in the Research Center for Care and Control of Infectious Diseases, Faculty of Medicine, Universitas Padjadjaran, Indonesia.

Mycobacterial Culture and DNA Extraction

Retrospective samples were retrieved and grown in Ogawa solid medium. After colonies emerged, total genome extraction was performed using the Cetyltrimethylammonium bromide (CTAB) based method adapted from Somerville et al in 2005.⁹ The sample was then put in 200 µL oTris-EDTA buffer and boiled at 85°C to kill the bacteria, followed by incubation in −20°C for 15 minutes and addition on 40 µL lysozyme (20 mg/mL). Following incubation in 37°C for 1 hour, the bacterial membrane and protein was broken down using SDS 1% and Proteinase K (250 µg/mL final concentration) with continuous agitation and incubation in 65°C. After incubation; the pellet and suspension phases were separated using 1% N-acetyl-N, N, N-trimethyl ammonium bromide (CTAB reagent) and NaCl. DNA was then precipitated with ethanol and eluted in 60 µL of nuclease-free water. The extracted DNA was stored at −20°C for other purposes.

PCR hsp65 Gene and DNA Sequencing

A conventional polymerase chain reaction (PCR) was performed on the DNA of 32 selected isolates. A mix consists of 1x PCR Master Mix Solution (HotStar Taq Master Mix Kit (Qiagen, Hilden, Germany), 0.3 µM of each forward-reverse hsp65 primers and 1.5 µL DNA template. The hsp65 primers used for the PCR (Forward: 5’- GGT CAA GGA AGT TGC CAA GA-3’ and reverse: 5’- ACC AGC AGG ATG TAG GGA TC-3’) were from in silico design. The PCR was performed with the following conditions: 95°C for 15 minutes, 35 cycles at 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 20 seconds and finalized at 72°C for 10 minutes. The amplicon visualization was performed using electrophoresis with 2% agarose gel. Negative (Nuclease-Free Water) and M. tuberculosis H37Rv controls were added in each reaction to detect cross reaction. The PCR product was then sequenced using the Sanger sequencing method with Applied Biosystems BigDye^® Terminator Cycle Sequencing Kit v3.1. The sequencing results were then analyzed, and gene profiling was carried out to determine the feasibility of the fragment as a DNA barcode. Sequence data was then imported in .abi and FASTQ files format, then trimming and base editing of the sequences results through alignment and adjustment with sequencing chromatograms using DNA Baser software (www.dnabaser.com). The results of sequence edits are saved in notepad format for later analysis for species identification. Sequence analysis and editing used DNA Baser software, while species identification used NCBI BLAST.

The study was approved by the Ethics Committee, Faculty of Medicine, Universitas Padjadjaran (434/UN6.KEP/EC/2020).

Results

A total of 2476 adult patients with suspected pulmonary TB were subjected to data screening; 1424 (57.5%) had positive culture results. Based on the screening of MPT64 antigen test results and formation of cord factor in the liquid culture, which differentiates between MTBC and NTM, 32 (2.2%) patients were determined as suspected NTM cases during the study period. Table 1 shows the demographic distribution of patients included in this study, with a majority being female (n = 19; 59.4%). The median age was 43.4 years old, with a higher distribution of patients aged 36–45 (28.1%). Some of the samples were collected from patients with previous history and treatment (40.6%). Only a few with HIV or DM (3.1% and 18.8%, respectively).

Table 1 Participant Sociodemographic Characteristics (N = 32)

The hsp65 gene fragments (446 bp) were successfully amplified for all the isolates. The sequence analysis identified some pathogenic NTM species (93.7%) and non-pathogenic species (6.3%) among 32 NTM isolates. M. abscessus (n = 13) and M. chelonae (n = 6) were the most prominent species identified in this study. Other pathogenic NTM species can also be found, such as members of the MAC complex (M. avium (n = 2), M. intracellulare (n = 1)), M. kansasii (n = 2), M. celatum (n = 2), and member of MFG (M. fortuitum (n = 2), M. arcueilense (n = 1), and M. conceptionense (n = 1)). The result also showed the identification of non-pathogenic NTM, M. vanbaaleni (n = 2). The highest prevalence from the sample population was M. abscessus (40.63%), followed by M. chelonae (18.75%), M. fortuitum group (12.51%), and MAC (M. avium complex) (9.38%) (Table 2).

Table 2 Identified NTM Species Based on hsp65 Partial Gene Sequencing

NTM speciation data can also be related to the type of colony growth, as well as patient data such as age, history of TB disease and DM status. NTM species were determined based on their presentation of similarity to the hsp65 reference sequence (Table 3).

Table 3 Identified NTM Species Based on hsp65 Partial Gene Sequencing, Looking into Subject’s Demographic and History Data

Discussion

NTM species known as opportunistic pathogen, which can cause human disease in complex clinical context, such as chronic pulmonary diseases. Giving symptoms which are similar to TB, NTM infections might be misdiagnosed as TB, or drug-resistant TB. Various factors, such as age, gender, HIV status, or history of chronic pulmonary diseases are associated with the susceptibility to NTM infection.¹⁰

As the burden of NTM infection among individual with presumptive TB cases is increasing, it is crucial to develop a reliable diagnostic approach for NTM disease and implement screening of NTM infection for presumptive TB patients with specific risk factors such as age and gender, and prior pulmonary disease.⁵ Furthermore, the development of tools to identifying NTM species will enhance our understanding of prevailing species and possible treatment given, as different NTM species require specific drug regimens, which may affect the effectiveness of therapy.

This study specifically focused on identifying NTM species from stored specimens obtained from individuals suspected of having NTM infections using partial hsp65 gene sequencing and discovering the prevalence of each identified NTM species in adult patients. The hsp65 gene, also known as the gro-EL2 gene, codes for a 65 KDa heat shock protein, which is present in all the species of mycobacteria.¹¹ This protein contains epitopes that are unique as well as common to various species of mycobacteria. While it has hypervariable regions which are very polymorphic, its conserved nature allows differentiation of mycobacteria using primers common to all mycobacteria.

The identification method for Mycobacterium species using the hsp65 gene was first implemented by Telenti et al.¹¹ At that time, the gene was used in a PCR pattern restriction enzyme analysis (PRA) method, employing a 441-bp fragment of the 1600-bp complete gene. Forty reference strains of Mycobacterium were used to establish a diagnostic algorithm. Later, the algorithm was applied to 290 clinical samples. Restriction enzyme digestion of those PCR products using BstEII and HaeIII resulted in the differentiation of Mycobacterium species based on their pattern of molecular size. This resulted identification of M. tuberculosis complex, M. gordonae, M. intracellulare, M. malmoense, M. avium, M. kansasii, M. szulgai, M. fortuitum complex, M. chelonae, and M. abscessus. Since then, the hsp65 gene has been very reliable in various Mycobacterium diagnostic methods.

In this research, a specifically designed primer set targeting the hsp65 partial gene was utilized, and it underwent the in-silico optimization step. In contrast to the primers which are widely used,¹¹ where the primer can still detect M. tuberculosis, the primer designed for this study excludes the M. tuberculosis complex, discriminate RGM and SGM, making it suitable for the specific identification of NTM. Our findings demonstrated that the hsp65 gene can unambiguously identify RGM species responsible for human infection, such as M. abscessus, M. chelonae, M. fortuitum, and M. peregrinum. The hsp65 gene offers an advantage over 16S rRNA gene sequencing in accurately identifying RGM species, which has been challenging to distinguish.¹²

The hsp65 gene is also effective in distinguishing different subsets within the SGM groups, such as the M. avium complex (MAC). Previous study used sequencing of hypervariable 3’ region of hsp65 to distinguish among subsets of MAC, which has been difficult to identify due to several factors, including the ambiguous phenotypic and genotypic assays as well as limitations in human and avian strains, which appear to be distinct.¹³ The obtained sequences indicate that M. intracellulare is genetically divergent from M. avium organisms, and distinct sequevars were identified for M. avium subsets, including M. avium subsp. avium (bird type), M. avium subsp. hominissuis, and M. avium subsp. paratuberculosis.

In this study, the hsp65 gene was selected as the target gene due to its presence in all Mycobacterium and has a considerable variation in its gene sequence, which has the potential to identify a bacterial species genetically. The hsp65 protein is also one of the most expressed Mycobacterium antigens, supported by a phylogenetic analysis which demonstrated the hsp65 gene capability to perform separation with a high level of confidence.^11,13

This study highlights the effectiveness of partial hsp65 gene sequencing in identifying various NTM species, which are known as opportunistic pathogens causing human disease. Of the 32 samples used in this study, M. abscessus was the most predominant species identified (40.63%). This finding is consistent with other previous studies, where M. abscessus was the most common rapidly growing mycobacteria identified from respiratory specimens.^14,15 A study conducted in Singapore found 49.9% of M. abscessus-chelonae group identified among 2026 NTM isolates.¹⁵ Similarly, M. abscessus was also predominant in a study conducted in Thailand, where 45 of 129 NTM specimen were identified as M. abscessus. ¹⁶

M. abscessus is a rapid-growing NTM pathogen that can cause lung disease, especially in susceptible patients with a history of cystic fibrosis, bronchiectasis, and tuberculosis.^17,18 It is also common to find M. abscessus in the environment, such as water and soil. At least 50% of cases of M. abscessus infection manifest as lung disease and after tuberculosis treatment,¹⁹ followed by M. chelonae (18.75%) and species of the MFG group (identified species consist of M. fortuitum (6.25%), M. arcueilense (3.13%) and M. conceptionense (3.13%)) which have also been well documented in the causation of NTM-PD. Moreover, recovered SGM species recovered, such as MAC (M. avium (6.25%), M. intracellulare (3.13%)) and M. kansasii (6.25%), have commonly been associated with pulmonary disease. Our research also discovered M. celatum (6.25%) and M. vanbaalenii (6.25%) among the NTM isolates. M. celatum, even though it is uncommon to cause human infection, can cause localized infections in the lungs and lymph nodes, mainly in immunocompromised hosts. In contrast, M. vanbaaleni is not known to be pathogenic.

Considering demographic and clinical history data, 10 out of 13 patients with a history of TB were suspected to be infected with RGM, and eight were identified as M. abscessus. This NTM species can cause pulmonary disease, especially in people with underlying structural lung disease and a history of tuberculosis.¹⁷ Although M. abscessus is considered as RGM species, this study found three out of 13 isolates identified as M. abscessus showed slow growth when were observed in liquid culture observation (Isolate 41101057, 41101072, 41100525, and 41100709). Those results are possibly correlated with each patient’s history of TB, as isolates of the M. abscessus complex, especially those from patients treated with multiple antimicrobial agents over time, often grow very slowly in liquid culture, even more than 5 days.²⁰ However in this study, the detail of each patient’s medication were not enclosed. Meanwhile, the risk factor for gender cannot be determined in this study because it requires a larger sample size.

In Indonesia, study related to NTM infections, particularly regarding species identification and the prevalence of each species, is limited. Previous research has been conducted in various settings with different research focuses and methods. Studies conducted Surakarta by Saptawati et al analyzed the 5-year laboratory data obtained from a lung hospital (Balai Besar Kesehatan Paru Masyarakat/BBKPM), Surakarta, Indonesia to calculate the prevalence of NTM and came up with a percentage of 15% among positive Mycobacterium culture, and 3.5% among suspected TB patients.⁶ Study from Mertaniasih et al, reported 5.78%of NTM infection positivity rate within TB suspect patients in Dr. Soetomo Hospital, Surabaya, Indonesia, which were diagnosed with niacin accumulation test and TB Ag MPT 64 (SD Bioline) test without providing species-specific information of each NTM isolates.^6,21, A study focusing on NTM identification had been done in Jakarta on analyzing 15 samples obtained from pulmonary specimens, skin, and soft tissues and showed the identification of M. abscessus, M. intracellulare, M. avium, and M. cookii by using 16S rRNA sequence analysis.²²

Despite being the second study conducted in Indonesia that applies molecular approach to identify NTM species, our research is the first study in Indonesia which demonstrates the use of partial hsp65 gene sequencing for the identification of NTM species and present the distribution of each species identified and expected to provide broader epidemiological data on NTM in the region, contributing to a more comprehensive understanding of the NTM prevalence among patients with pulmonary infection. To expand our understanding, further studies about species identification using sequence-based analysis are needed, preferably with a larger sample size that covers a wider geographic area of Indonesia. The low proportion of NTM found in our study reflects the study subjects (study population) which were recruited from primary health care facilities with a high likelihood of being TB disease. The findings might be different in the hospitals or lung clinics where patients come with more severe disease and have risk factors for NTM infection. Conducting a prospective cohort study embedded with TB cohort would be ideal to t to maximize the benefits for the patients.

Conclusion

In conclusion, partial hsp65 gene sequencing has a high discriminatory power to identify NTM species in specimens from Indonesia. This research successfully identified several significant NTM species known to be opportunistic pathogen through sequence-based analysis of the hsp65 gene. M. abscessus became the most prevalent species among the NTM specimens analyzed in this study, followed by M. chelonae, M. fortuitum group (MFG), M. avium complex (MAC), M. celatum, M. vanbaalenii. The identification of NTM species can help clinicians in selecting appropriate treatment strategies, reducing the risk of misdiagnosis with TB. Based on our findings, partial hsp65 gene sequencing holds a potential to serve as an appropriate first-line identification method for NTM isolated from human samples.